This document discusses chromatography and key concepts in the chromatographic process. It explains that in chromatography, a sample is introduced and the solute molecules distribute between the stationary and mobile phases. The distribution causes separation of compounds over time as the mobile phase moves through the column. It also defines important terms like retention time, hold up time, peak width, and asymmetry factor which are used to characterize chromatographic separation and peaks. The document explores how differences in the distribution coefficient between substances allows their separation in the column.

1. Lecture two
ChromatographyChromatography
Analytical Chemistry 4
Second stage
Dr.Ashraf Saad Rasheed

2. The Chromatographic Process
• A chromatographic separation is illustrated in Figure 1.1.
A sample is introduced at the beginning of theA sample is introduced at the beginning of the
chromatographic column.
• The solute molecules are distributed between the
stationary and mobile phases according to an appropriate
distribution constant As the mobile phase moves thedistribution constant. As the mobile phase moves, the
compounds become separated.
•
2 Dr.Ashraf Saad Rasheed

3. Figure 1.1: The chromatographic process
3 Dr.Ashraf Saad Rasheed

4. • Physical principles of chromatographic separation
Retention parametersp
• In a chromatographic method, we observe a dynamic
fequilibrium for analytes between the phases involved.
• This balance is the equilibrium distribution A successful• This balance is the equilibrium distribution. A successful
separation is given only if the distribution coefficient DA of
the substances to be separated is sufficiently different.
• DA is defined as the ratio of the concentration of a substance
(A) between mobile ( ) and stationary phase ( ) Substances(A) between mobile (M) and stationary phase (S). Substances
with a high distribution coefficient DA are more strongly
retained by the stationary phase than those with small
distribution coefficients.
4 Dr.Ashraf Saad Rasheed

5. tR2
tR1
R2
tM
eighth
ntensity
1,000
0,882
0 607
σ
2σ
ntensity
tS1
tS2
Injection
Peakhe
Signalin
0,607
0,500
0,134
2σ
Wh = 2,354σ
4σa b
10 %
Wb
Wh
Signalin
Tim e Peak widthPeak width
10 %
Wb = 4 σ
( )a ( )b ( )c
( ) d l d h (b) f f hFigure 1.2: (a) Idealized chromatogram. (b) Definition of the
asymmetry factor.
(c)
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( )
Gaussian distribution with characteristic parameters.

6. • In a chromatographic column, two different analytes areg p , y
separated if they spend different times in the stationary
phase as in Figure 1.2 (a).
Th ti f th t i d l t t i• The time necessary for the non-retained analytes to move is
called the hold-up time tM, also sometimes referred to as
dead time or void time.
• The analyte retention time tS is defined as the time for
solutes not to move along the column. As in Equation 1.2,
the gross retention time or residence time t of analytes onthe gross retention time or residence time tR of analytes on
the stationary phase is obtained from the analyte retention
time and column hold-up time:
6 Dr.Ashraf Saad Rasheed

7. tR2
tR1
R2
tM
eighth
ntensity
1,000
0,882
0 607
σ
2σ
ntensity
tS1
tS2
Injection
Peakhe
Signalin
0,607
0,500
0,134
2σ
Wh = 2,354σ
4σa b
10 %
Wb
Wh
Signalin
Tim e Peak widthPeak width
10 %
Wb = 4 σ
( )a ( )b ( )c
Fi 1 2 ( ) Id li d h (b) D fi i i f hFigure 1.2: (a) Idealized chromatogram. (b) Definition of the
asymmetry factor.
(c)( )
Gaussian distribution with characteristic parameters.
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8. •The retention volume VR is calculated from the solute
retention time and a constant of flow rate of mobile phase F:retention time and a constant of flow rate of mobile phase F:
•The asymmetry factor AS is defined as the ratio of the
distances (tail portion b and front portion a) between thedistances (tail portion-b and front portion-a) between the
central verticals and the slopes of the distribution at 10% of
their height as shown in Figure 1.2 (b).
•As in Equation 1.4, the asymmetry factor AS is calculated of
peak distortion:p
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9. tR1
tR2
th
sity
1,000
0,882 σ
sity
tS1
tS2
tM
Peakheight
Signalintens
0,607
0,500
2σ
Wh = 2,354σWh
Signalintens
Tim e
Injection
Peak widthPeak width
0,134 4σa b
10 %
Wb = 4 σ
( )a ( )b ( )c
Wb
Figure 1.2: (a) Idealized chromatogram. (b) Definition of the
asymmetry factor.
(c)
Gaussian distribution with characteristic parameters.
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10. • For asymmetry factors > 1, the asymmetry is called
tailing Tailing effects occur via a fast increase of thetailing. Tailing effects occur via a fast increase of the
chromatographic signal followed by a relatively slow
decrease; primarily responsible for this effect are
adsorption processes.
• For asymmetry factors < 1 the asymmetry is called• For asymmetry factors < 1, the asymmetry is called
fronting. The image of the peak shape of this effect is the
opposite of the tailing, fronting effect which happens if
the stationary phase does not have a sufficient number
of suitable adsorption sites.
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